This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We recently identified a class of small molecules named "cotransins" that inactivate a subset of secreted and transmembrane proteins by blocking their cotranslational translocation into the endoplasmic reticulum. The N-terminal signal sequence of the secreted or transmembrane protein is sufficient to confer sensitivity or resistance to cotransin. However the molecular basis for this selectivity is unknown. Utilizing a cotransin photo-affinity probe, we identified Sec61alpha, a ten-pass integral membrane protein that forms the structural core of the Sec61 translocation channel, as the direct target of cotransin. Sec61 forms the channel through which all secreted and membrane proteins traverse during cotranslational translocation. We hypothesize that cotransin blocks translocation by stabilizing a closed conformation of the channel and by preventing a productive interaction between Sec61 and the translocating protein substrate. We propose to precisely map the photo-crosslinking site of cotransin using mass spectrometry. These studies are designed to decipher the precise mechanism by which cotransin inhibits cotranslational translocation and the molecular basis for its remarkable substrate selectivity.